Assessment of a 16S Rrna Amplicon Illumina Sequencing Procedure for Studying the Microbiome of a Symbiont-Rich Aphid Genus
Molecular Ecology Resources (2016) 16, 628–640 doi: 10.1111/1755-0998.12478 Assessment of a 16S rRNA amplicon Illumina sequencing procedure for studying the microbiome of a symbiont-rich aphid genus E. JOUSSELIN,* A.-L. CLAMENS,* M. GALAN,* M. BERNARD,† S. MAMAN,‡ B. GSCHLOESSL,* G. DUPORT,§ A. S. MESEGUER,* F. CALEVRO§ and A. COEUR D’ACIER* *INRA – UMR 1062 CBGP (INRA, IRD, CIRAD, Montpellier SupAgro), 755 avenue du Campus Agropolis CS 30016, F-34 988 Montferrier-sur-Lez, France, †INRA – UMR 1313 GABI–SIGENAE, INRA de Jouy en Josas, Domaine de Vilvert, 78352 Jouy en Josas, France, ‡INRA, GenPhySE, Sigenae, Chemin de Borde rouge -CS 52627, 31326 Castanet Tolosan, France, §UMR 203 BF2I, Biologie Fonctionnelle Insectes et Interactions, INRA, INSA de Lyon, Universite de Lyon, 20 Avenue Einstein, F-69621 Villeurbanne, France Abstract The bacterial communities inhabiting arthropods are generally dominated by a few endosymbionts that play an important role in the ecology of their hosts. Rather than comparing bacterial species richness across samples, ecologi- cal studies on arthropod endosymbionts often seek to identify the main bacterial strains associated with each speci- men studied. The filtering out of contaminants from the results and the accurate taxonomic assignment of sequences are therefore crucial in arthropod microbiome studies. We aimed here to validate an Illumina 16S rRNA gene sequencing protocol and analytical pipeline for investigating endosymbiotic bacteria associated with aphids. Using replicate DNA samples from 12 species (Aphididae: Lachninae, Cinara) and several controls, we removed individual sequences not meeting a minimum threshold number of reads in each sample and carried out taxonomic assignment for the remaining sequences.
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